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Band 3, the major anion transport protein of human erythrocytes, forms the core of a multiprotein complex in the erythrocyte membrane. Here we studied the spatiotemporal mechanisms of band 3 multiprotein complex assembly during erythropoiesis. Significant pools of intracellular band 3 and Rh-associated glycoprotein (RhAG) were found in the basophilic erythroblast. These intracellular pools decreased in the polychromatic erythroblast, whereas surface expression increased and were lowest in the orthochromatic erythroblast and reticulocytes. Protease treatment of intact cells to remove extracellular epitopes recognized by antibodies to band 3 and RhAG was used to study surface delivery kinetics and intracellular complex composition from the proerythroblast stage to the enucleated reticulocyte. Newly synthesized band 3 and protein 4.2 interact initially in the early stages of the secretory pathway and are found associated at the plasma membrane from the basophilic stage of erythropoiesis. Although we could successfully coimmunoprecipitate Rh with RhAG from plasma membrane pools at a similar stage, no intracellular interaction between these proteins was detectable. Knockdown of RhAG during early erythropoiesis was accompanied by a concomitant drop in membrane expression of Rh polypeptides. These data are consistent with assembly of major components of the band 3 macrocomplex at an early stage during erythropoiesis.

The blood group Rh antigens are associated with non-glycosylated 30-kDa erythrocyte membrane proteins (the Rh30 polypeptides) and the Rh glycoprotein. We used antipeptide antibodies to study the Rh glycoprotein in human erythrocyte membranes. The Rh glycoprotein was present in Rhnull U+ve cells. However, the N-glycan chain of the Rh glycoprotein in Rhnull U+ve cells was smaller than in normal cells. In contrast, the N-glycan chain of the Rh glycoprotein was larger than normal in glycophorin B-deficient red cells. We suggest that this observation reflects a lower rate of movement of newly synthesized Rh glycoprotein through intracellular membranes to the cell surface in the absence of glycophorin B, and that in normal red cells glycophorin B facilitates the movement of the Rh protein complex to the cell surface. Our results provide evidence for the intracellular interaction of at least three proteins, the Rh glycoprotein, Rh30 polypeptides, and glycophorin B during the biosynthesis and cell surface expression of the Rh complex. These observations are likely to be important for the successful design of expression systems for the blood group Rh antigens.

The Rh blood-group antigens are associated with human erythrocyte membrane proteins of approx. 30 kDa (the Rh30 polypeptides). Heterogeneously glycosylated membrane proteins of 50 and 45 kDa (the Rh50 glycoproteins) are coprecipitated with the Rh30 polypeptides on immunoprecipitation with anti-Rh-specific mono- and poly-clonal antibodies. We have isolated cDNA clones representing a member of the Rh50 glycoprotein family (the Rh50A glycoprotein). We used PCR with degenerate primers based on the N-terminal amino acid sequence of the Rh50 glycoproteins and human genomic DNA as a template and cloned and sequenced three types of PCR product of the expected size. Two of these products, Rh50A and Rh50B, gave the same translated amino acid sequence which corresponded to the expected Rh50 glycoprotein sequence but had only 75% DNA sequence similarity. The third product (Rh50C) contained a single base deletion, and the translated amino acid sequence contained an in-frame stop codon. We have isolated cDNA clones containing the full coding sequence of the Rh50A glycoprotein. This sequence predicts that it is a 409-amino acid N-glycosylated membrane protein with up to 12 transmembrane domains. The Rh50A glycoprotein shows clear similarity to the Rh30A protein in both amino acid sequence and predicted topology. Our results are consistent with the Rh30 and Rh50 groups of proteins being different subunits of an oligomeric complex which is likely to have a transport or channel function in the erythrocyte membrane. We mapped the Rh50A gene to human chromosome 6p21-qter, showing that genetic differences in the Rh30 rather than the Rh50 genes specify the major polymorphic forms of the Rh antigens.

The Rh-blood-group antigens (often described as Rhesus antigens) are associated with erythrocyte membrane proteins of approx. 30 kDa. We have determined the N-terminal 54 amino acid residues of the 30 kDa Rh D polypeptide (D30 polypeptide). We used primers based on these sequence data and the polymerase chain reaction (PCR) on human reticulocyte cDNA and genomic DNA to clone two types of PCR product of identical size. The two PCR products had related translated amino acid sequences between the 3' ends of the primers, one of which was identical with that found for the D30 polypeptide. We designate the two related mRNA species which gave rise to the PCR products as Rh30A and Rh30B, the latter corresponding to the D30 polypeptide. We have isolated cDNA clones for the Rh30A protein which encode a hydrophobic membrane protein of 417 amino acids. The Rh30A protein has the same N-terminal 41 amino acids as the D30 polypeptide, but beyond this point the sequence differs, but is clearly related. The Rh30A protein probably corresponds to the R6A32 polypeptide, another member of the Rh 30 kDa family of proteins, which may carry the C/c and/or E/e antigens. Hydropathy analysis suggests that the Rh30A protein has up to 12 transmembrane domains. Three of these domains are bordered by a novel cysteine-containing motif, which might signal substitutions at these cysteine residues. Information which supplements this paper (amino-acid-sequence-analysis histograms) is reported in Supplementary Publication SUP 50160 (4 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1990) 265, 5.

The human red cell membrane components reacting with monoclonal antibody MB-2D10 were examined by immunoblotting. The antibody bound to a diffusely staining band extending from Mr 30,000 up to the high-molecular-weight region of the gel in normal membranes and in Rhnull U + membranes, but not in Rhnull U - membranes. Treatment of normal red cells with an endoglycosidase F-containing preparation destroyed the epitope recognized by MB-2D10. The reactivity of the antibody with purified preparations of Rh-related glycoproteins D30 polypeptide, D50 polypeptide, R6A32 polypeptide, and R6A45 polypeptide was also examined. Only the purified R6A45 and D50 components reacted with MB-2D10. These results show that MB-2D10 recognizes a carbohydrate-dependent epitope on the R6A45 and D50 group of Rh-related polypeptides. The results also suggest the possibility that the U antigen arises from interaction between glycophorin B and the Rh-related components D50 and R6A45.

Cytoskeleton preparations derived from lactoperoxidase-radioiodinated human erythrocytes were found to be enriched in a labelled component with the same apparent molecular mass as the Rhesus (D) (Rh(D] antigen polypeptide. Immune precipitation from the cytoskeleton preparations confirmed that this component is the Rh(D) polypeptide. The results suggest that the Rh(D) polypeptide may be linked to the erythrocyte skeletal matrix. The possibility that the Rh(D) antigen is involved in maintaining the shape and viability of the erythrocyte is discussed.

Rhnull human erythrocytes lack all the antigens of the Rhesus blood-group system and are associated with mild chronic haemolytic anaemia. These erythrocytes have an abnormal shape and increased osmotic fragility. Labelling studies with the impermeant maleimide N-maleoylmethionine [35S]sulphone show that Rhnull erythrocytes lack two extracellular thiol-group-containing membrane components of apparent mol.wts. 32 000 and 34 000. Immunoprecipitation with mouse monoclonal antibody R6A (which reacts with all normal erythrocytes, but fails to react with Rhnull erythrocytes) specifically precipitates the 34 000-mol.wt. component from normal erythrocytes. Similar studies with human anti-Rh(D) serum shows that this antibody reacts with the 32 000-mol.wt. component. The results suggest that the R6A-binding polypeptide and the Rh(D) polypeptide may be involved in the maintenance of the shape and viability of the human erythrocyte.

Immunoprecipitation using a monoclonal antibody showed that the Wrb antigen is present on the abnormal (delta-alpha) hybrid sialoglycoprotein of Sta-positive human erythrocytes but not on the abnormal (delta-alpha) hybrid sialoglycoprotein of Dantu-positive erythrocytes. These results provide further information regarding the nature and location of the Wrb antigen on the normal erythrocyte sialoglycoprotein alpha.

Membranes from erythrocytes with a new Gerbich (Ge)-negative phenotype (Leach phenotype), as well as those from two other Ge-negative phenotypes, were examined. Whereas cells of the Leach phenotype apparently lack three minor sialoglycoproteins (beta, beta 1 and gamma), the membranes of Ge- Yus- and Ge- Yus+ erythrocytes apparently lack beta- and gamma-sialoglycoproteins but contain additional diffusely migrating components of apparent Mr 30 500-34 500 and 32 500-36 500 respectively. Immunoprecipitation experiments showed that the abnormal components of both Ge- Yus- and Ge- Yus+ erythrocytes reacted with two monoclonal antibodies, BRIC 4 and BRIC 10. These antibodies have been shown to react with sialoglycoproteins beta and beta 1 in normal erythrocytes. Cytoskeletal preparations from Ge- Yus- and Ge- Yus+ erythrocyte membranes contained the abnormal components. In contrast with cells of the Leach phenotype, which are elliptocytic, Ge- Yus- and Ge- Yus+ were of normal shape, despite their apparent lack of beta- and gamma-sialoglycoproteins. It seems likely that the abnormal components in these cells contribute to their normal shape. Ovalocytic erythrocytes were shown to incorporate more radioactivity in the sialoglycoprotein-beta 1 region than normal erythrocytes after labelling by the periodate/NaB3H4 technique. It is suggested that abnormal components in Ge- Yus- and Ge- Yus+ erythrocytes result from chromosomal misalignment with unequal crossing-over at meiosis between the genes giving rise to beta-, beta 1- and gamma-sialoglycoproteins.

1. Immunoprecipitation of periodate/NaB3H4-labelled human erythrocytes using anti-Wrightb (Wrb) monoclonal antibodies showed that these antibodies specifically react with the major erythrocyte sialoglycoprotein alpha (glycophorin A). 2. Similar experiments on erythrocytes from the only known individual lacking the Wrb antigen but with otherwise normal sialoglycoproteins did not result in the immunoprecipitation of any sialoglycoprotein. 3. We suggest that the Wrb antigen is located on an alpha-helical region between residues 55 and 70 of sialoglycoprotein alpha.